Hematopoietic cell culture or ex vivo expansion of the number of hematopoietic cells is a rapidly growing area of tissue engineering with many potential applications in bone marrow transplantation, gene therapy and production of blood platelets etc. In recent years the number of allogenic bone marrow transplants have increased considerably as they have become a therapeutic option for hematologic, immunologic and malignant disorders. In addition, it has become a clinical practice to perform bone marrow transplants following chemotherapy and radiotherapy in cancer patients because chemotherapy and radiotherapy cause damage to the hematopoietic progenitors in the bone marrow and render the patient susceptible to infection.
At present the process of bone marrow transplantation is associated with various complications and discomfort. Bone marrow harvesting is painful and requires use of operating room procedures. Large numbers of cells are required for transplant and the early treatment relies on the infusion of a sufficient number of progenitor cells. Moreover compatible donors are in short supply. As a result, there has been much interest in the expansion of the number of bone marrow cells ex vivo prior to transplantation. Ex vivo expansion of hematopoietic cells has the potential to decrease the initial harvest necessary for successful engraftment. Through ex vivo cell expansion techniques, a small marrow specimen taken under local anaesthetic can be expanded into the large number of cells required for transplantation, thereby eliminating the large harvest procedure. Engraftment may be accelerated by increasing the number of progenitor and immature cells available for infusion. In addition, it may be possible to cryopreserve expanded cells to be infused at multiple time points, thereby allowing multiple cycles of chemotherapy. The use of expanded cells may also allow increased doses of chemotherapy thereby facilitating tumor reduction. More importantly, ex vivo expansion of the number of bone marrow cells has the potential for improving the transplant outcome by allowing clinicians to transplant more cells, to supplement transplants with mature progenitors to speed the recovery of neutrophils and platelets and to use a single hematopoietic harvest for repeated transplants over an extended period. Similar procedures could also be used for treatment of AIDs patients where HIV infected leukocytes could be eliminated by chemotherapy/radiotherapy and the autologous bone marrow cells expanded ex vivo could be transplanted back to restore the immunocompetence. The method for ex vivo expansion of the number of hemopoietic cells could also be used to generate activated and antigen sensitized immunocompetent cells for immunotherapy of cancer and infections. In addition, such methods may also be used for ex vivo expansion of genetically transfected or transformed hematopoietic cells for gene therapy.
The method of ex vivo expansion of the number of hematopoietic cells involves taking a small quantity of bone marrow or hematopoietic stem cells and producing more outside the body for later transplantation. Since the demonstration that direct contact between primitive hematopoietic cells and stroma is not required for long term in vitro hematopoiesis (Verfaillie, 1992) growth factors and cytokines have been used to substitute for the supportive function of the adherent layer (Zandstra et al. 1994). This has led to the development of methods for supporting bone marrow cell growth involving the use of cytokines, especially IL-6 and IL-3, and growth factors like stem cell factor (SCF) in the liquid media (Bernstein et al., 1991). IL-6 has been shown to act synergistically with IL-3 and SCF to augment the proliferation of human hematopoietic progenitor cells and to support colony formation from dormant murine hematopoietic progenitors (Ikebuchi et al., 1987: Koike et al.; 1988, Tanaka et al.; 1992). Cytokines and growth factors have been used both for static as well as perfusion cultures. Much greater expansion of the number of colony-forming cells is achieved in the presence of cytokines, IL-6 and IL-3 and SCF in perfusion bioreactor than in static culture (Koller et al.; 1993 a,b). Other culture systems used for the scale-up of marrow cultures include suspension, micro carriers, airlift and hollow fiber bioreactors.
Although there are various methods available for the ex vivo expansion of the number of cells using various combinations of cytokines or stromal cells, the magnitude of expanded progenitor cells that has been achieved, especially multi potential progenitors, is typically low. Moreover, the requirement for use of cytokines and growth factors whether natural or recombinant make the currently used methods very expensive and therefore more cost effective methods are needed urgently.
The method of ex vivo expansion of the number of hematopoietic cells described in this application, uses a water soluble plant extract prepared from a plant of the Tinospora species, belonging to family Menispermacease, preferably the plant is Tinospora cordifolia. Plants of the Tinospora species have been widely used in traditional Indian medicine for treatment of skin infections, arthritis, fever, dysentery, urinary tract infections, and diabetes (Gupta et al., 1967; Sharma and Sharma, 1981; Raghunathan and Sharma, 1969). Tinospora cordifolia has also been reported to protect mice against E. coli induced abdominal sepsis (Thatte et al., 1987, Thatte and Dahanukar, 1989). Crude preparations of Tinospora cordofolia have been used in the preparation of herbal formulations for treatment of skin diseases (Shah, U.S. Pat. No. 5,693,327, 1997), diabetes, (Dhaliwal, U.S. Pat. No. 5,886,029, 1997) and arthritis (Chavali et al., U.S. Pat. No. 5,683,698, 1997). None of these reports and patents suggest or describe the use of this plant material as in this invention.